Role of dysregulated miRNA in Tregs in Multiple Sclerosis

描述（申请人提供）：多发性硬化症（MS）是一种免疫介导的中枢神经系统（CNS）脱髓鞘疾病，可导致严重的神经功能障碍。该疾病的病因尚不清楚，该疾病病理生理学背后的免疫和神经退行性机制尚不清楚，目前的治疗方法仅在减缓疾病进展方面部分有效。我的实验室对未经治疗的MS患者的初始和效应/记忆CD4 T细胞进行了大规模的miRNA分析研究，以确定miRNA是否可能有助于疾病的易感性或进展。预测TGF¿信号通路在MS患者中被CD4 T细胞中被鉴定为失调的12个mirna抑制。TGF¿已被证明在CD4+CD25hiFoxp3+调节性T细胞（Tregs）中起着至关重要的作用，而Tregs已被证明在MS患者中存在缺陷。TGF¿信号已被证明对iTregs的产生和维持treg中的抑制功能和Foxp3表达至关重要。我们假设MS患者CD4 T细胞中miRNA表达异常导致TGF信号受损和CD4调节性T细胞缺陷，从而促进MS的发生和进展。目的1：确定MS患者CD4 T细胞中miRNA表达异常是否直接靶向TGF信号通路中的特定分子。首先，我们将确定MS患者和健康对照者CD4 T细胞中特异性mirna与预测TGF¿靶点之间的基因表达水平是否存在相关性。其次，我们将使用荧光素酶测定来确定特异性mirna是否抑制TGF¿通路中预测靶基因的3' UTR序列。目的2：确定靶向TGF -通路的mirna是否改变treg的发育和/或抑制treg的功能。TGF -靶向mirna在初始CD4 T细胞中被抑制或过表达，然后在iTreg条件下培养。由此产生的T细胞将表型为iTreg特征。此外，将用特异性miRNA抑制剂转染Tregs，评估其对TGF¿的敏感性和抑制效应T细胞的能力。目的3：确定TGF -相关miRNA表达水平与体内Tregs数量之间是否存在关联，或者TGF -相关miRNA表达与MS患者Tregs抑制能力之间是否存在关联。流式细胞术分析MS患者外周血CD4 T细胞将评估MS患者中Tregs的数量与TGF¿-相关mirna的表达。此外，我们将从MS患者外周血中对Treg进行分类，评估其抑制能力，并确定TGF¿-相关mirna与Treg数量和/或功能之间是否存在相关性。鉴于Treg在维持对自身耐受性方面的作用已被充分证明，并且观察到MS患者中CD4 Treg存在缺陷，这项新研究将确定CD4 T细胞中miRNA表达失调是否导致MS患者的Treg缺陷。虽然有几项研究已经确定了在Tregs17-22中发挥作用的mirna，但没有研究发现MS患者中与TG通路中的基因特异性相关的差异表达mirna及其对自身免疫性疾病中Tregs的影响。Treg细胞在保护我们的环境和保护我们自己免受自身免疫系统的微妙平衡中发挥着重要作用，TGF¿具有超越Treg的多营养作用，对健康和疾病具有巨大的重要性，使这项研究成为一项宝贵的研究。

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS) which can result in severe neurological deficits. The cause of the disease is unknown, the immune and neurodegenerative mechanisms underlying the pathophysiology of the disease are poorly understood, and current therapies are only partially effective at slowing disease progression. My laboratory has performed a large miRNA profiling study on naive and effector/memory CD4 T cell in untreated MS patients to determine if miRNA may contribute to disease susceptibility or progression. The TGF¿ signaling pathway was predicted to be suppressed in MS patients by 12 miRNAs identified as being dysregulated in CD4 T cells. TGF¿ has shown to play a vital role in CD4+CD25hiFoxp3+ regulatory T cells (Tregs), and Tregs have been shown to be defective in MS patients. TGF¿ signaling has been shown to be critical for production of iTregs, and the maintenance of suppressor functions and Foxp3 expression in Tregs. We hypothesize that dysregulated miRNA expression in CD4 T cells in MS patients' results in impaired TGF¿ signaling and defective CD4 regulatory T cells, promoting the onset and progression of MS. Aim 1: To determine if miRNAs identified as dysregulated in CD4 T cells in MS patients directly target specific molecules in the TGF ¿ signaling pathway. First, we will determine if there is a correlation in gene expression levels between specific miRNAs and predicted TGF¿ targets in human CD4 T cells from MS patients and healthy controls. Second, we will use luciferase assays to determine if specific miRNAs suppress the 3' UTR sequence of the predicted target genes in the TGF¿ pathway. Aim 2: To determine if the miRNAs that target the TGF¿ pathway alter iTreg development and/or suppressive functions of Tregs. TGF¿ targeting miRNAs will be inhibited or over-expressed in naive CD4 T cells and then cultured under iTreg conditions. The resulting T cells will be phenotyped for iTreg characteristics. In addition, Tregs will be transfected with specific miRNA inhibitors, evaluated for sensitivity to TGF¿, and ability to suppress effector T cells. Aim 3: To determine if there is an association between the level of TGF¿-associated miRNA expression and the number of Tregs in vivo, or an association between TGF¿-associated miRNA expression and suppressive capacity of Tregs in MS patients. Flow cytometric analysis of peripheral blood CD4 T cells from MS patients will evaluate the number of Tregs in MS patients relative to the expression of TGF¿-associated miRNAs. In addition, Tregs will be sorted from peripheral blood of MS patients, evaluated for suppressive capacity, and determine if there is a correlation between TGF¿-associated miRNAs and Treg number and/or function. Given the well-documented role of Tregs in maintaining tolerance to self and the observation that CD4 Tregs are defective in MS patients, this novel study will determine if dysregulated miRNA expression in CD4 T cells underlies this Treg defect in MS patients. Although several studies have identified miRNAs that play a role in Tregs17-22, no studies have identified differentially expressed miRNAs in MS patients that are specifically associated with genes in the TG¿ pathway and their influence on Tregs in an autoimmune disease. Treg cells play an important role in the delicate balance of protection from our environment and protecting ourselves from our own immune system, and TGF¿ has pleiotrophic effects that extend beyond Tregs that have vast importance in health and disease, making this an invaluable study.